While the traditional focus of basic and clinical cancer immunology research has been on MHC class I-restricted cytolytic CD8+ T lymphocytes, our laboratory has innovated investigations into the role of MHC class II-restricted CD4+ T cells in human antitumor immunity. CD4+ cells can be crucial for initiating, orchestrating and maintaining antitumor immune responses in animal models. This laboratory has been on the forefront of developing new technologies for the biochemical and molecular identification of tumor-associated proteins recognized by CD4+ T cells, including protein purification and cDNA library screening approaches targeting the MHC class II Ag processing/presentation pathway. Current projects include:1) Clinical and laboratory investigations using the melanoma-associated Ag tyrosinase (Ty) as an immunogen. We previously demonstrated that Ty, an enzyme catalyzing melanin synthesis and expressed in metastatic melanoma lesions, could be targeted by both CD4+ and CD8+ T cells. Poxvirus vectors encoding Ty have been used in conjunction with systemic interleukin-2 in two NCI protocols, to treat patients with stage IV metastatic melanoma. As part of these efforts, a major initiative has been to develop new in vitro methods for monitoring immune reactivities to whole proteins such as Ty. Recombinant adenovirus was used to express Ty in autologous dendritic cells, which were then used to stimulate fresh autologous PBL from vaccinated patients transiently in vitro. T cell activation was assessed with real time RT-PCR measuring cytokine mRNA expression, demonstrating the generation of Ty-specific T cells following vaccination in select patients. A current project aims to develop a recombinant lentiviral vector for this purpose, offering the advantages of Ag expression in several different kinds of Ag presenting cells without encoding extraneous immunogenic viral proteins. Such methods may also be useful for the efficient evaluation of "candidate" tumor Ag identified through genomic or proteomic screening, and present an attractive alternative to the cumbersome peptide library approaches currently employed for this purpose. 2) Evaluation of novel tumor Ag for clinical application. Following the recent finding that a single missense mutation in the signaling molecule BRAF is associated with >60% of melanomas, our laboratory has investigated whether mutant BRAF can be recognized by CD4+ T cells from melanoma patients. T cells specific for a mutant 29-mer peptide, restricted by a variety of HLA-DR, -DP, and -DQ alleles, have been generated from 4 melanoma patients harboring the commonly expressed V599E mutation. CD4+ T cells from two patients restricted by HLA-DRB10404 were also shown to recognize whole melnaoma cells expressing the restricting HLA allele and the BRAF mutation. Additional melanoma patients, as well as MHC class I-restricted immunity, will be studied as a prelude to possible clinical development of immunotherapies targeting mutant BRAF. The V599E mutation has also been reported in 35% of papillary thyroid cancers and 10% of colon cancers, offering the possibility of clinical application in other cancer histologites. Mutant BRAF is a tumor-associated Ag whose expression may be vital for initiating or maintaining the mailignant phenotype. As such, it exemplifies tumor Ag that may be optimal targets for immunotherapy, since the in vivo seleciton of Ag loss variants is unlikely to occur.